Three Rs Approaches in the Quality Control of Inactivated Rabies Vaccines

The Report and Recommendations of
ECVAM Workshop 481,2

Reprinted with minor amendments from ATLA 31: 429-454.

Appendix 1

Best Practice Guide for the Batch Potency Testing of Inactivated Rabies Vaccine Using Mice

Introduction

ECVAM workshop report 48 includes a number of recommendations on the application of the Three Rs in the quality control of inactivated rabies vaccines (1). With regard to the possibilities of reduction, two important aspects, which significantly reduce the overall number of animals, are repeated here: the grouping of controls, which leads to reduction when the same control groups are used for back titration of the challenge virus and for the reference vaccine; and the introduction of a single-dilution test, provided that it has been validated in the laboratory (2).

The current methods used for the potency testing of inactivated rabies vaccines for human and veterinary use are based on the intracerebral (i.e.) challenge of mice that have previously been vaccinated intraperitoneally. This guideline is not meant as a complete protocol for rabies vaccine potency testing, but focuses on techniques which should be applied in order to refine the animal procedure, thus minimising the suffering of the animals.

The following preconditions should be met:

L.c. injection into mice is stipulated also for other pharmacopoeial tests (for example, the pertussis vaccine potency test [31 or extraneous agents testing [4, 5]), and is used for diagnostic purposes (for example, for the diagnosis of rabies [6]).

In the following paragraphs, step-by-step guidance is given for the necessary animal procedures, taking into account all possibilities for refinement of the techniques applied.

 

Vaccination of Mice

Groups of animals are vaccinated with different dilutions of the test vaccine. Each group is kept in a box or in several boxes, depending on the number of animals required. The animals in a group should be individually marked for identification; small spots of picric acid, applied at different sites of the body, are suitable for this purpose. Vaccination is performed by intraperitoneal injection of the vaccine, using a 25ga x 5/8 needle, as illustrated in Figures 1 and 2. This procedure is not painful, and anaesthesia of the mice is therefore not necessary.

Figure 1: Catching and immobilisation of the mouse

a)

b)

a) Catching, b) immobilisation.

Figure 2: Intraperitoneal vaccination

l.c. Challenge with Virulent Rabies Virus

The i.c. challenge should be carried out only on anaesthetised animals, since it involves considerable pain and might induce shock. The technique applied should permit the anaesthetisation of the animals in their boxes and should avoid the risk of mixing animals between groups; moreover, it should be rapid and safe.

Anaesthetisation technique

Gaseous anaesthesia (for example, with isoflurane, or halothane) is routinely used, as it is necessary to have a rapid induction with a recovery time of 10-20 minutes. Premedication with an analgesic should not be performed, since the effects on the outcome of the test are not known.

Furthermore, it should be possible to anaesthetise mice in their boxes. For example, a gaseous system with a home-made box in which several cages can be installed, could be used. Among the products available, halothane has a "wake-up time" of more than 10 minutes, which allows the removal of the anaesthetisation device from the box, and the inoculation of all the mice. A trained person will need less than 2 minutes to inoculate ten mice.

I.c. injection procedure

In order to reduce the risk of contamination for the person carrying out the inoculation, the mouse should be immobilised in the abdominal position, as shown in Figures 3 and 4. The syringe (World Health Organization: 0.25-0.5 ml tuberculin syringe; 7) should be held vertically, and the needle should point toward the plate and not toward the fingers of the person carrying out the inoculation. A 27ga (8) needle, which is either sheathed or very short, as shown in Figure 5, should be used to permit penetration of not more than 5mm. Modifications concerning the position for the i.e. injection were described by Koprowski (9).

Figure 3: Injection site

Figure 4: Immobilisation of the mouse in the abdominal position, and intracerebral inoculation

a)

b)

a) Immobilisation; b) intracerebral inoculation.

Figure 5: Needles for intracerebral inoculation

Humane endpoints

The i.e. injection of even small volumes is likely to cause brain damage, even if it is performed properly. The two European Pharmacopoeia monographs on rabies vaccines for human and veterinary use (10, 11) consider death occurring before day 5 after challenge to be non-specific and not attributable to rabies infection. Therefore, it is necessary to carefully observe the animals after recovery from anaesthesia. Adverse reactions can range from sudden death to mild ataxia or circling to the traumatised side. Animals showing neurological signs during the first days after infection should be excluded from the experiment and humanely killed.

The frequency of inoculation-induced adverse reactions should be carefully monitored. The use of score sheets is recommended for the documentation of clinical signs.

As has been underlined in the conclusions and recommendations of the ECVAM workshop report (1), it is possible to use humane endpoints and euthanise the mice as soon as they exhibit clinical signs of rabies, without reducing the sensitivity of the potency test (see 12). However, an exception should be made: in order to maintain the virulence of the challenge virus strain, it is passaged in mice. In this case, the animals should only be humanely killed when they are paralysed; thus, a higher virus titre can be achieved, and fewer animals will be used for the preparation of a challenge virus strain batch.

 

References

  1. Bruckner, L., Cussler, K., Haider, M., Barrat, J., Castle, P., Duchow, K., Gatewood, D.M., Gibert, R., Groen, J., Knapp, B., Levis, R., Milne, C., Parker, S., Stünkel, K., Visser, N. & Volkers, P. (2003). Three Rs approaches in the quality control of inactivated rabies vaccines. ATLA 31, 429-445.
  2. Aubert, M.F.A. & Blancou, J. (1982). Test simplifié de contrôle d'activité des vaccins antirabiques à virus inactivés. Revue Scientifique et Technique Office International des Epizooties 1, 811-822.
  3. Council of Europe (2002). Assay of pertussis vaccine (2.7.7.). In The European Pharmacopoeia, 4th edn, pp. 169-170. Strasbourg, France: Council of Europe.
  4. Council of Europe (2002). Tests for extraneous agents in viral vaccines for human use (2.6.16.). In The European Pharmacopoeia, 4th edn, pp. 148-149. Strasbourg, France: Council of Europe.
  5. Council of Europe (2002). Cell substrates for the production of vaccines for human use (5.2.3.). In The European Pharmacopoeia, 4th edn, pp. 415-418. Strasbourg, France: Council of Europe.
  6. OIE (2000). Rabies. In Manual of Standards for Diagnostic Tests and Vaccines, 4th edn, pp. 276-291. Paris, France: Office International des Epizooties. Web site http://www.oie.int/eng/normes/ mmanual/A_summry.htm (Accessed 11.8.03).
  7. Wilbur, L.A. & Aubert, M.F.A. (1996). The NIH test for potency. In Laboratory Techniques in Rabies, 4th edn (ed. FX. Meslin, M.M. Kaplan & H. Koprowski), pp. 360-368. Geneva, Switzerland: World Health Organization.
  8. USDA (1999). Supplemental Assay Method for Potency Testing of Inactivated Rabies Vaccine in Mice Using the National Institutes of Health Test; MVSAM0308.01, 19pp. Ames, IA, USA: USDA, APHIS.
  9. Koprowski, H. (1996). The mouse inoculation test. In Laboratory Techniques in Rabies, 4th edn (ed. F-X. Meslin, M.M. Kaplan & H. Koprowski), pp. 80-87. Geneva, Switzerland: World Health Organization.
  10. Council of Europe (2002). Rabies vaccine for human use prepared in cell cultures (Monograph No. 0216). In The European Pharmacopoeia, 4th edn, pp. 2213-2214. Strasbourg, France: Council of Europe.
  11. Council of Europe (2002). Rabies vaccine (inactivated) for veterinary use (Monograph No. 0451). In The European Pharmacopoeia, 4th edn, pp. 2282-2284. Strasbourg, France: Council of Europe.
  12. Anon. (2003). The use of humane endpoints in the quality control of rabies vaccines. ATLA 31, 450-454.

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